Oligonucleotides are widely utilized in molecular biological manipulations including DNA sequencing, in vitro mutagenesis, cloning methodologies involving polylinkers and adapters, synthesis of genes by hybridization and ligation of multiple oligonucleotides, and the like methods. Traditionally, oligonucleotides are prepared by chemical synthesis methods de novo each time they are required.
For DNA sequencing, unique oligonucleotide sequencing primers are required as each new sequence is identified. Chemical synthesis of oligonucleotides is time consuming and custom synthesis is costly.
Recently, Studier proposed a strategy to simplify the preparation of unique oligonucleotides in the form of a library of pre-synthesized oligonucleotides representing every possible nucleotide sequence in the size range of oligonucleotides from 8 to 10 nucleotides in length. Studier, Proc.Natl.Acad.Sci.USA, 86:6917-6921 (1989). The library poses technical difficulties insofar as the library must contain from 4.sup.8 (65,536) to 4.sup.10 (1,048,576) members, respectively, which is generally considered to be so large as to be unmanageable.
Szybalski proposed the use of a library of hexameric oligonucleotides comprising every possible combination of nucleotide bases, representing a library having 4.sup.6 (4,096) members. Szybalski, Gene, 90:177-178 (1990). Theoretically, the hexamers in the library were proposed to be capable of being individually ligated to form 12 nucleotide (nt), 18-nt, or 24-nt oligonucleotides in length, therefore forming every possible nucleotide sequence from a library having 4,096 members.
However, it has not been shown that hexamers can actually be used to form 12-nt long sequencing primers.